DNA Sequencing Application in Crop Sciences

Gene Sequencing: The process of determining the sequence of nucleotides in a segment of DNA. It uses any method or technology that can be used to determine the sequence of DNA bases: adenine, guanine, thymine, and cytosine. Gene sequencing and genetic engineering are utilized to create new types of crops that are very important today and in the future. Incorporating crop genetics into farming practice has a massive influence on the efficiency and quality of life of crop agriculture. With the ability to apply DNA sequencing to crop science, farmers and scientists alike can increase crop variety and handpick certain desirable genetic traits that can apply to other crops.

According to Gincy Paily Thottathil in her article Sequencing Crop Genomes: A Gateway to Improve Tropical Agriculture, “DNA sequence information is extremely valuable for identifying key genes controlling important agronomic traits and for identifying genetic variability among the cultivars.” In the same article, it is said that many vital crops have already been sequenced and have had their key traits recorded for further studies. These crops include cereals, tuber crops, vegetables, and fruits. With the ongoing development of DNA sequencing, society has a possibility of seeing a second green revolution due to the limitless potential of this technology and practice.

Introduction

In most places around the world, the land is too dry, cold, steep, and salty for farming; these conditions make it difficult for people to grow crops and force us to only use about 11% of Earth’s lands (about 1.5 billion hectares in comparison to 13.4 billion hectares). Additionally, our biggest problem is the large, growing population because we have yet to find a complete, efficient, and sustainable way to feed the growing amount of people. However, people have used next-plant genome sequencing to aid this difficult situation; this has resulted in many of the crops that are used to sustain most of the population today.

For many years, studies that use DNA re-sequencing and gene expression have been performed to substantially improve our understanding of crop genetics. Our newfound information about crop genomes, through these studies, has been applied to develop and enhance many crop varieties. The applications of DNA sequencing technologies will continue to improve in the coming future and will continue to aid in much-needed crop improvements.

Timeline of release of genome sequences for key crop species. (source)

In terms of genetically modified crops, many have been created to aid undeveloped and poor regions of the world. Some of these crops include vegetables, fruits, and oil plants. Functional markers have been developed for many of these crops, and genes controlling agronomically essential traits have been identified. However, re-sequencing and gene expression studies continue to be completed to comprehensively understand the genetic mechanism behind each trait and identify variations. For example, rice (Oryza sativa) has become one of the most important crops in these countries. During the 1990s, people took an interest in improving this crop.

Specifically, in 1997, the International Rice Genome Sequencing Project was formed, consisting of 11 countries. These countries began to look at the 12 chromosomes of Oryza sativa to try and find useful information. After 7 years of work, they were able to gather that “the genome size was found to be 389 Mb, comprising 37,544 protein-coding genes” (Thottathil).

Features of the Rubber Tree Genome.

(A) Circular representation of the 18 pseudochromosomes; (B) the density of genes; (C) the density of non-coding RNA; (D) the distribution of transposable elements (TEs); (E) the distribution of gypsy-type retrotransposons; (F) the distribution of copia-type retrotransposons; (G) the distribution of DNA transposons; (H) SSR density; (I) the distribution of GC content; (J) whole-genome duplication (WGD) event shown by syntenic relationships among duplication blocks containing more than 15 paralogous gene pairs. (source)

Eventually, this information led to the development of varieties of rice with “improved yield, high nutritional quality, and improved tolerance towards diseases, pests, different soil conditions, and stresses such as drought and flood” (Thottathil). Even though “Golden Rice” was created in the 1990s (before the International Rice Genome Sequencing Project), it is a perfect example of DNA sequencing. The project’s goal was to distribute O. sativa to the 11 participating countries for genome analysis using NGS.

Although the data varied from each country, the information obtained from the project proved indispensable for rice genomics’s future. “These data were used to elucidate a major QTL for rice grain production, Gn1a, which was later identified as a cytokinin oxidase/dehydrogenase, an enzyme that degrades cytokinin” (Ashikari et al. 2005). Furthermore, the transcription factor controlling Gn1a was also identified as a zinc finger transcription factor which has been “reported to regulate drought and salt tolerance in rice” (Huang et al. 2009b).

This is a type of rice species that was created as a Vitamin A supplement. People inserted the genes of daffodils, soil bacteria, or corn as a way to create a nutritional supplement for people in poorer and more underdeveloped regions (Golden Rice photo from www.isaaa.org). In addition to food crops, a few other economically important crops were also sequenced. People are desperately trying to improve these crops because they are the main source of income for many poor countries. One such example is natural rubber, which comes from the tree Hevea brasiliensis.

Basic flow chart of the CRISPR/Cas9 genome editing system.

The engineered CRISPR/Cas9 system consist of two components; (1a) the Cas9 endonuclease and, (1b) a single-guide RNA (sgRNA). “The sgRNA contains a spacer sequence followed by 79 nt of an artificially fused tracrRNA and crRNA sequence”, (2) The spacer sequence is typically 20 nt in length, and specifically binds to the target DNA sequence containing a 5’-NGG-3’ PAM motif at the 3’ end, which is highly specific for the gene of interest, (3) The fused trans-activating crRNA (tracrRNA) and crRNA sequence forms a stem-loop RNA structure that binds to the Cas9 enzyme; tracrRNA hybridizes and joins Cas9. (4) Assembly of sgRNA, attached with the target sequence and the Cas9 vector construct. (5) Transformation of the vector construct into rice via different transformation techniques. (5a) Screening and selection of rice mutant plants based on phenotypic changes. (5b) Restriction enzyme site loss generating a CRISPR/Cas9 mutagenized plant line. (c, control; m, mutagenized; RE, restrictions enzyme). (5c) Surveyor Assay (CEL1 and T7 are DNA endonucleases utilized in surveyor assay). (5d) Next-generation sequencing. (6) Future analysis to obtain T-DNA-free plants, and further experiments to prove phenotypic changes cast by the knockout of the gene under investigation. (source)

With next-generation sequencing technologies, several transcriptome sequencing projects have been completed for the Hevea brasiliensis species. Through these projects, we have found that “approximately 78% of the genome was repetitive DNA” (Thottathil). Additionally, a “total of 68,955 gene models were predicted, of which 12.7% are unique to [Hevea brasiliensis]” (Thottathil). We have been able to identify important genes of the species like which ones are associated with rubber synthesis and disease resistance. The genomic information provided a good foundation for crop improvement of this species; this crop now has an increased yield and less susceptibility to diseases because of this valuable information. (Hevea brasiliensis picture from keys.lucidcentral.org) One of the most practiced methods of agriculture is plant breeding. Applying DNA sequencing to crop science can allow farmers and scientists to identify and exploit genetic variations in other plants. “Identification of the key genes underlying a trait enables the transfer of the trait to another cultivar or species by genetic modification; alternatively, these traits may be incorporated into a cultivar by marker-assisted selection” (Edwards & Batley 2010). A number of crops have been sequenced (Table 1), and the data we have obtained from them can help immensely with the cultivation of more crops in the future.

Features of major sequenced crop genomes. (source)

Futuristic view

On November 15, 2022, the human population reached 8 billion people. Now, there are over 800 million people who are malnourished and undernourished, acute food insecurity has raised to 345 million from 135 million just 3 years ago, and 49 countries are on the edge of having a famine. We already struggle to provide the food needed for our current number, but our population continues to grow at a fast rate. We are also battling against climate change, which continuously makes productive agriculture difficult due to the harsher climates. Soil erosion is another problem that plagues many areas around the world. This worsens the health of many rivers and streams, we are constantly losing fertile land that could be used to grow more food for other people, and people often turn valuable wetlands into agricultural lands as a way to “fix” our problems but this worsens the state of the Earth and climate change. This is why people need to start using sustainable agriculture, a way we can support the people while not compromising future generations, that uses genetically modified crops.

GMOs that have built-in pest resistance can reduce the number of pesticides that we use today; however, we need to create a variety that won’t be susceptible to insects that will become immune to these crops. Additionally, farmers overuse fertilizer to make sure their plants grow well; however, this leads to excessive runoff, which pollutes and leads to eutrophic bodies of water. We could sequence plants that use fewer nutrients to grow to significantly lower the amount of fertilizer used as a way to solve this problem.

Finally, we need to sequence easy-to-grow crop varieties that can aid the malnourished population. People need food that will continue to grow in harsh climates and give them the nutrients they need daily. This will significantly help people that are struggling with overpopulation and are living in underdeveloped countries. Although there is much room for improvement involving DNA sequencing, the implications of the process are limitless, and future utilization can provide a pathway for a second green revolution which could potentially save billions of lives worldwide.

Article By:

• Brandon Phan (High School)

• Clair Kim (High School)

  (External students, post on the request of Mentors)

References​

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